Production of tumor angiogenesis factor by cell culture

ABSTRACT

Process for the production of human TAF in vitro comprising growing the human osteosarcoma cell line MG-63 in agitated, liquid suspension of nutrient culture medium at about 35°-38° C. for a sufficient time to elaborate TAF and recovering the resulting TAF from the cells or cell product.

BACKGROUND OF THE INVENTION

This invention relates to the in vitro production of human tumorangiogenesis factor from the human osteosarcoma cell line MG-63.

It has been known for some time that unless a solid tumor is providedwith blood vessels by its host it remains small and dormant. Thesubstance that is released by tumors and provides vascularization hasbeen named tumor angiogenesis factor (TAF) by Dr. Judah Folkman of theHarvard Medical School. J. Exptl. Med. 133, 275-88 (1971); Cancer Res.34, 2109-13 (1974).

Ever since it was recognized that tumor induced neovascularizationrepresents the breaching of an important barrier in the control of tumorgrowth, considerable effort has been spent in searching for ways toinhibit neovascularization. It was early suggested by Dr. Folkman thatblockade of this factor (inhibition of angiogenesis) might arrest solidtumors at a tiny diameter of a few millimeters (avascular phase). NewEngl. J. Med. 285, 1182 (1971); J. Exptl. Med. 133, 275-88 (1971). Onesuggested approach was the raising of antibodies against TAF extractsfor the production of an anti-serum. Folkman, Ann Surg. 175, 409-16(1972); Phillips et al., Int. J. Cancer 17, 549-58 (1976). Anotherapproach to inhibiting neovascularization which has received muchattention recently consists in treatment of tumor cells with extracts ofcartilage. Eisenstein et al, Am. J. Pathol. 73, 765-74 (1973); Sorgenteet al., Lab. Invest. 32, 217-22 (1975); Eisenstein et al., Amer. J.Pathol. 81, 337-48 (1975); "Medical News", JAMA 232 (1), 14-15 (1975).;Brem and Folkman, J. Exptl. Med. 141, 427-39 (1975); and Kuettner etal., U.S. Pat. No. 4,042,457. These efforts are limited, of course, tothe amount of TAF which is available and suitable for test purposes.

TAF finds further use in the development of tests such as an angiogenicassay or a diagnostic screening test for neoplasia. Klasbrun et al,Cancer Res. 36, 110-14 (1976); and Brem et al., Science 195, 880-81(1977); Cancer 41, 239-44 (1978).

TAF also has been proposed as useful for wound healing. Rettusa et al,FASEB, Abstract No. 4309, 61st Ann. Meet., Apr. 1-8, 1977, Chicago, Ill.

Various human tumor cells have been reported heretofore as capable ofelaborating TAF when measured by certain assays. Thus, extracts fromhuman neuroblastoma, Wilms' tumor and human hepatoblastoma were found tocontain TAF which was able to cause the formation of new blood vesselsin the subcutaneous fascia of rats. Folkman et al., J. Exptl. Med. 133,275-88 (1971).

So also, WI-38 embryonic lung SV W126 (SV 40 virus transformed W126),glioblastoma, meningioma and HeLa cells (the latter only in suspensionculture and not in monolayers) grown in T-75 tissue culture flasks weredescribed as giving a positive TAF response as measured by bioassay onthe chick chorioallantoic membrane (CAM). Folkman and Klagsbrun, Chapter31 of "Fundamental Aspects of Neoplasia", at page 402 (Gottlieb et al,eds.), Springer-Verlag, N.Y., 1975.

Subsequently, extracts from hypernephroma, haemangioma and human kidneywere similarly described as eliciting a TAF response which resulted inthe growth of new capillaries in the subcutaneous fascia of rats.Phillips et al., Int. J. Cancer 17, 549-58 (1976).

In still other experiments, Hubler and Wolf demonstrated TAF responsesfrom human cutaneous melanoma implanted in transparenthamster-cheek-pouch membranes. Cancer 38, 187-92 (1976).

All of the foregoing tumor cells are derived from fresh tumors orprimary cultures except the WI-38, SV W126 and HeLa cells. Fresh tumorsand primary cultures are not, however generally suitable sources of TAFexcept for limited research purposes or small scale production. In orderto provide a commercially significant source of TAF in terms of readyavailability and adequate supply, production from a suitable establishedcell line is deemed necessary. As a practical matter the cell lineshould have not only specific TAF activity, but it should also have goodcell growth characteristics in terms of rapid growth, good suspensiongrowth, adaptability to large-scale culture and economical nutrientrequirements.

The terms "cell line" and "established cell line" are used herein inconformance with the definitions published by Federoff in the TissueCulture Association Manual, Vol. 1, No. 1, pp. 53-57 (1975).

DESCRIPTION OF THE INVENTION

Applicants have investigated numerous human tumor cell lines for theproduction of TAF, but most of them have been eliminated as unsuitablecandidates in view of their poor growth characteristics asabove-defined.

One cell line that has now been found to have good growthcharacteristics is suspension culture and is able to elaborate thedesired TAF in suitable quantities is the human osteosarcoma cell lineMG-63. The established cell line MG-63 was derived from an osteosarcomaby A. Billiau as reported by Billiau et al., Antimicrob. AgentsChemother. 12 (1), 11-15 (1977). Cultures of this cell line areavailable from A. Billiau, Rega Institute for Medical Research, Leuven,Belgium, and from the American Type Culture Collection, Rockville, Md.,under the code designation ATCC CRL 1427. Although this cell line isdescribed as useful for the production of interferon, it has notheretofore been known as a suitable source of TAF.

Initially, the medium used by applicants for maintenance and growth ofthis cell line was Dulbecco's modification of Eagle's minimum essentialmedium (MEM) containing 4 mg/ml of glucose. The culture medium wassupplemented with 15% by volume fetal bovine serum without addition ofany antibiotics, although lower concentrations of fetal bovine serum,for example, about 10-15%, also can be used. The cells were first grownat 37° C. in attached culture in 75 cm² T-flasks (Falcon Plastics) andwere then converted to agitated, liquid suspension culture in the samemedium in 4-liter and 100-liter vessels in accordance with the proceduredescribed in U.S. Pat. No. 4,059,485. The cells were then harvestedafter 7 to 12 days growth since inoculation, and TAF was extracted andassayed according to the general procedures described in U.S. Pat. No.4,059,486.

Specifically, the cells after growth were washed successively threetimes in lactated Ringer's solution, resuspended in phosphate bufferedsaline (pH 7-7.4) and stirred for 4 hours at 4° C. The supernatant afterremoval of the cells by centrifugation was retained as the cell extract.The cells pellet from the extract was resuspended in distilled water andstirred at 37° C. for 20 minutes. The supernatant after removal of thecell debris by centrifugation was retained as the cell lysate.

In various runs of 4 liters, and 100-110 liters the cell extracts andcell lysates were assayed to contain the following amount of totalprotein (Lowry protein assay):

    ______________________________________                                               Suspension                                                                              Time Since                                                          Volume    Inoculation                                                                              Total                                             Run No.                                                                              (Liters)  (Days)     Protein                                           ______________________________________                                        1       4        12         24.7   mg.   extract                                                          99.8   mg.   lysate                               2       4        10         8.4    mg.   extract                                                          116    mg.   lysate                               3      110        7         0.66   gm.   extract                                                          2.5    gm.   lysate                               4      110        7         0.52   gm.   extract                                                          1.0    gn.   lysate                               ______________________________________                                    

The cell extracts from the 4-liter runs gave strongly positive tests inthe bioassay for TAF in the chorioallontoic membrane (CAM) of chickembryos, in accordance with the procedure described by Folkman, CancerRes. 34, 2109-13; 36, 110-14 (1976).

The cell extracts from the 100- to 110-liter runs were subjected tofurther purification by CM-Sephadex® (Pharmacia) chromatography andlyophilized. The purified material also gave a positive test in the CAMbioassay for TAF. The use of CM-Sephadex chromatography for obtaining aTAF active fraction from tumor cells is described by Tuan et al.,Biochemistry 12 (17), 3159-65 (1973).

It will be appreciated that other nutrient culture media for culture ofMG-63 cells can be used in place of Dulbecco's MEM, for example, any ofthe well-known tissue culture media described by H. J. Morton, In Vitro6, 89-108 (1970). These conventional culture media contain knownessential amino acids, mineral salts, vitamins and carbohydrates. Theyare also frequently fortified with mammalian sera such as fetal bovineserum. Suitable growth of the cells can be carried out at about 35°-38°C. but cell proliferation is best at 37° C. Growth under these cellculture conditions for about 4-8 days generally is sufficient to producethe desired TAF.

Other suitable equipment and procedures for growing cells in agitated,liquid suspension which can be adapted for culture of the cell lineMG-63 will be readily apparent to the person skilled in the art byreference to well-known texts on cell culture such as, for example,Paul, "Cell and Tissue Culture," The Williams and Wilkins Company,Baltimore, 4th ed. 1970, at pages 277-291; Kruse and Patterson, "TissueCulture Methods and Applications," Academic Press, New York, 1973, atpages 333-377.

It should also be understood that various means can be used to separateand purify TAF-containing fractions from the cells and cell cultureproduct other than the illustrative specific recovery proceduresdescribed above. These various recovery means include the knowntechniques for the separation and purification of proteinaceoussubstances in general such as, for example, dialysis, salt and solventprecipitation, adsorption with gels, cellulose ion exchangechromatography, Sephadex gel filtration, electrophoresis, andlyophilization. Thus, TAF can be obtained by extraction from the cellsfollowed by subjecting the extract to dialysis against NaCl (0.15 M),Sephadex G-100 chromatography, dialysis against water and lyophilizationanalogous to the methods described by Folkman et al., J. Exptl. Med.133, 275-88 (1971) and Phillips et al., Int. J. Cancer 17, 549-58(1976). According to Phillips et al., id, when the dialysate issubjected to chromatography on a column of Sephadex G-100 in 0.15 MNaCl, the fractions between 35,000 and 300,000 molecular weight containthe greatest TAF activity. According to Folkman, Cancer Res. 34, 2109-13(1974), TAF is a proteinaceous substance having a molecular weight ofapproximately 100,000. It will be appreciated, however, that theaforesaid molecular weights are estimates based on partially purifiedTAF-containing products and the inventors are not bound by suchestimates or by any particular purification procedure or TAF purity.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. All such further examples are includedwithin the scope of the appended claims.

What is claimed is:
 1. Process for the production of human TAF in vitrocomprising growing the human osteosarcoma cell line MG-63 in agitated,liquid suspension of nutrient culture medium at about 35°-38° C. for asufficient time to elaborate TAF and isolating the resulting TAF fromthe cells or cell product.
 2. The process of claim 1 in which thenutrient culture medium is Dulbecco's modification of Eagle's minimumessential medium.
 3. The process of claim 1 in which the nutrientculture medium is fortified with mammalian serum.
 4. The process ofclaim 1 in which the nutrient culture medium is fortified with fromabout 10% to about 15% fetal bovine serum.
 5. The process of claim 1 inwhich the TAF is isolated by extraction from the cells.
 6. The processof claim 1 in which the nutrient culture medium is Dulbecco'smodification of Eagle's minimum essential medium and is fortified withfetal bovine serum and in which the TAF is isolated by extraction fromthe cells.